Increased survival of neonatal pigs by supplementing medium-chain triglycerides in late-gestating sow diets

Two experiments were conducted to study the efficacy and causes of medium-chain triglycerides (MCT) in sow diet in improving the survival of neonatal pigs. In Experiment 1, beginning on d84 of gestation and continuing through d28 of lactation, 51 sows were fed corn–soybean meal diet mixed with either soybean oil (SO; n=17), coconut oil (CO; n=18), or MCT (n=16) in a proportion of 9 : 1 by weight. The highest improvement in survival of pigs by sows fed MCT (p<0.01) or CO (p<0.05) was observed during the first three days after birth in pigs weighing <1100 g at birth, compared with sows fed SO. Their three-day survival was 98.6, 80.0 and 47.6%, respectively, for MCT, CO and SO groups. In Experiment 2, beginning on d84 of gestation and continuing through farrowing, 24 sows, 8 sows per treatment, were fed diets as in Experiment 1. Liver glycogen content of pigs 4 h after born from sows fed MCT (p<0.10) and CO (p<0.01), and muscle glycogen of pigs from sows fed MCT (p<0.01) and CO (p<0.10) were increased, compared to those of pigs from sows fed SO. Plasma albumin was increased by MCT and CO (p<0.01), relative to SO. The results suggest that MCT or CO in sow diets may enhance the body glycogen stores and maturity of pigs at birth and, hence, their survival, particularly in pigs with low birth weight during the first three days of life.     

Okadaic acid stimulates carnitine palmitoyltransferase I activity and palmitate oxidation in isolated rat hepatocytes

Okadaic acid parallely increased carnitine [corrected] palmitoyltransferase I activity and the rate of palmitate oxidation in isolated rat hepatocytes. Nevertheless, okadaic acid had no significant effect on the rate of octanoate oxidation. Maximal effects of okadaic acid were similar and non-additive to those of dibutyryl-cAMP, forskolin and glucagon. Results indicate that carnitine palmitoyltransferase I activity may be controlled by a mechanism of phosphorylation/dephosphorylation.     

The liver isoform of carnitine palmitoyltransferase I is activated in neonatal rat cardiac myocytes by hypoxia

Fatty acids are the preferred substrate of ischemic, reperfused myocardium and may account for the decreased cardiac efficiency during aerobic recovery. Neonatal cardiac myocytes in culture respond to hypoxia/serum- and glucose-free medium by a slow decline in ATP which reverses upon oxygenation. This model was employed to examine whether carnitine palmitoyltransferase I (CPT-I) modulates high rates of -oxidation following oxygen deprivation. After 5 h of hypoxia, ATP levels decline to 30% control values and CPT- I activity is significantly stimulated in hypoxic myocytes with no alteration in cellular carnitine content or in the release of the mitochondrial matrix marker, citrate synthase. This stimulation was attributed to an increase in the affinity of hypoxic CPT-I for carnitine, suggesting that the liver CPT-I isoform is more dominant following hypoxia. However, there was no alteration in hypoxic CPT-I inhibition by malonyl-CoA. DNP-etomoxiryl-CoA, a specific inhibitor of the liver CPT-I isoform, uncovered identical Michaelis kinetics of the muscle isoform in control and hypoxic myocytes with activation of the liver isoform. Northern blotting did not reveal any change in the relative abundance of mRNA for the liver vs. the muscle CPT-I isoforms. The tyrosine phosphatase inhibitor, pervanadate, reversed the hypoxia-induced activation of CPT-I and returned the affinity of cardiac CPT-I for carnitine to control. Reoxygenation was also associated with a return of CPT-I activity to control levels. The data demonstrate that CPT-I is activated upon ATP depletion. Lower enzyme activities are present in control and reoxygenated cells where ATP is abundant or when phosphatases are inhibited. This is the first suggestion that phosphorylation may modulate the activity of the liver CPT-I isoform in heart.     

Stereoisomeric acylamidomorpholinium carnitine analogues: selective inhibitors of carnitine palmitoyltransferase I and II

Acylamidomorpholinium carnitine analogues, 6-(tetradecanamidomethyl- and -hexadecanamidomethyl)-4,4-dimethylmorpholin-4-ium-2-a cetate, 1, synthesized as complete sets of stereoisomers, were assayed as inhibitors for isozymes of carnitine palmitoyltransferase (CPT). Microsomal CPT isoymes showed modest discrimination among the stereoisomers; while rat-liver mitochondrial CPT-I and CPT-II showed distinct differences. The tetradecanamidomethyl analogue of (2R,6S)-1 activated CPT-I but inhibited CPT-II.     

Induction of peroxisomal beta-oxidation enzymes in primary cultured rat hepatocytes by clofibric acid

Rat hepatocytes were cultured for 72 h with or without the addition of 0.5 mM clofibric acid. The activities of individual enzymes of the peroxisomal beta-oxidation pathway (acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and 3-ketoacyl-CoA thiolase) decreased in the control culture, but markedly increased synchronously in the clofibric acid-treated culture. The levels of mRNAs coding for these enzymes and the rates of synthesis of the enzymes were also elevated in the clofibric acid-treated culture, although no proportional relationship was observed between the time-dependent changes of these parameters. The increase in mRNAs was much higher than the increase in the rate of synthesis of the enzymes. The activity of catalase, its mRNA level and the rate of its synthesis were slightly affected. The effects of clofibric acid on the peroxisomal beta-oxidation enzymes and catalase in primary cultured hepatocytes were very similar to those observed in vivo. These results, therefore, suggest that primary culture of hepatocytes should provide a useful means for investigating the mechanism of induction of peroxisomal enzymes and the mechanism of action of peroxisome proliferators.     

Differential induction of peroxisomal beta-oxidation enzymes by clofibric acid and aspirin in piglet tissues

Peroxisomal beta-oxidation (POX) of fatty acids is important in lipid catabolism and thermogenesis. To investigate the effects of peroxisome proliferators on peroxisomal and mitochondrial beta-oxidation in piglet tissues, newborn pigs (1-2 days old) were allowed ad libitum access to milk replacer supplemented with 0.5% clofibric acid (CA) or 1% aspirin for 14 days. CA increased ratios of liver weight to body weight (P < 0.07), kidney weight to body weight (P < 0.05), and heart weight to body weight (P < 0.001). Aspirin decreased daily food intake and final body weight but increased the ratio of heart weight to body weight (P < 0.01). In liver, activities of POX, fatty acyl-CoA oxidase (FAO), total carnitine palmitoyltransferase (CPT), and catalase were 2.7-, 2.2-, 1.5-fold, and 33% greater, respectively, for pigs given CA than for control pigs. In heart, these variables were 2.2-, 4.1-, 1.9-, and 1.8-fold greater, respectively, for pigs given CA than for control pigs. CA did not change these variables in either kidney or muscle, except that CPT activity was increased approximately 110% (P < 0.01) in kidney. Aspirin increased only hepatic FAO and CPT activities. Northern blot analysis revealed that CA increased the abundance of catalase mRNA in heart by approximately 2.2-fold. We conclude that 1) POX and CPT in newborn pigs can be induced by peroxisomal proliferators with tissue specificity and 2) the relatively smaller induction of POX in piglets (compared with that in young or adult rodents) may be related to either age or species differences.     

Importance of acyl-CoA availability in interpretation of carnitine palmitoyltransferase I kinetics

Bovine serum albumin is generally employed as a substrate depot for the delivery of acyl units to lipid metabolizing enzymes in vitro. Here we test the possibility that albumin alters the availability of substrate to mitochondrial carnitine palmitoyltransferase I and thereby alters its apparent kinetics. Binding competition with palmitoyl-CoA indicates that albumin has 5-6 high affinity sites which avidly bind the substrate, while isolated mitochondria compete favorably for substrate only as the albumin sites become saturated. In contrast to albumin, artificial phospholipid vesicles bind palmitoyl-CoA uniformly. Palmitoyl-CoA distribution between vesicles and mitochondrial membranes appears simply to be a function of the relative size of the two lipid compartments. Both albumin and artificial vesicles reduce the effective concentration of substrate available to the enzyme and in this way reduce apparent affinity. Direct measurement of mitochondrially bound substrate removes this effect and brings the results into agreement with an affinity constant of 6-7 nmol/mg. Changes in gross mitochondrial structure, as indicated by decreased optical density and increased nonpelleting protein, do not begin occurring until levels of mitochondrially bound palmitoyl-CoA are 15 times greater than this. The highly sigmoidal activity profile of carnitine palmitoyltransferase with respect to palmitoyl-CoA (apparent Hill coefficient = 3.0 +/- 0.3) is lost when vesicles are substituted for albumin, suggesting that albumin binding sites contribute to the sigmoidal kinetics in the range of palmitoyl-CoA studied.     

Interaction of malonyl-CoA and 2-tetradecylglycidyl-CoA with mitochondrial carnitine palmitoyltransferase I

Malonyl-CoA and 2-tetradecylglycidyl-CoA (TG-CoA) are potent inhibitors of mitochondrial carnitine palmitoyltransferase I (EC 2.3.1.21). To gain insight into their mode of action, the effects of both agents on mitochondria from rat liver and skeletal muscle were examined before and after membrane disruption with octylglucoside or digitonin. Pretreatment of intact mitochondria with TG-CoA caused almost total suppression of carnitine palmitoyltransferase I, with concomitant loss in malonyl-CoA binding capacity. However, subsequent membrane solubilization with octylglucoside resulted in high and equal carnitine palmitoyltransferase activity from control and TG-CoA pretreated mitochondria; neither solubilized preparation showed sensitivity to malonyl-CoA or TG-CoA. Upon removal of the detergent by dialysis the bulk of carnitine palmitoyltransferase was reincorporated into membrane vesicles, but the reinserted enzyme remained insensitive to both inhibitors. Carnitine palmitoyltransferase containing vesicles failed to bind malonyl-CoA. With increasing concentrations of digitonin, release of carnitine palmitoyltransferase paralleled disruption of the inner mitochondrial membrane, as reflected by the appearance of matrix enzymes in the soluble fraction. The profile of enzyme release was identical in control and TG-CoA pretreated mitochondria even though carnitine palmitoyltransferase I had been initially suppressed in the latter. Similar results were obtained when animals were treated with 2-tetradecylglycidate prior to the preparation of liver mitochondria. We conclude that malonyl-CoA and TG-CoA interact reversibly and irreversibly, respectively, with a common site on the mitochondrial (inner) membrane and that occupancy of this site causes inhibition of carnitine palmitoyltransferase I, but not of carnitine palmitoyltransferase II. Assuming that octylglucoside and digitonin do not selectively inactivate carnitine palmitoyltransferase I, the data suggest that both malonyl-CoA and TG-CoA interact with a regulatory locus that is closely juxtaposed to but distinct from the active site of the membrane-bound enzyme.     

Overexpression of carnitine palmitoyltransferase I in skeletal muscle in vivo increases fatty acid oxidation and reduces triacylglycerol esterification

A key regulatory point in the control of fatty acid (FA) oxidation is thought to be transport of FAs across the mitochondrial membrane by carnitine palmitoyltransferase I (CPT I). To investigate the role of CPT I in FA metabolism, we used in vivo electrotransfer (IVE) to locally overexpress CPT I in muscle of rodents. A vector expressing the human muscle isoform of CPT I was electrotransferred into the right lateral muscles of the distal hindlimb [tibialis cranialis (TC) and extensor digitorum longus (EDL)] of rats, and a control vector expressing GFP was electrotransferred into the left muscles. Initial studies showed that CPT I protein expression peaked 7 days after IVE (+104%, P<0.01). This was associated with an increase in maximal CPT I activity (+30%, P < 0.001) and a similar increase in palmitoyl-CoA oxidation (+24%; P<0.001) in isolated mitochondria from the TC. Importantly, oxidation of the medium-chain FA octanoyl-CoA and CPT I sensitivity to inhibition by malonyl-CoA were not altered by CPT I overexpression. FA oxidation in isolated EDL muscle strips was increased with CPT I overexpression (+28%, P<0.01), whereas FA incorporation into the muscle triacylglycerol (TAG) pool was reduced (-17%, P<0.01). As a result, intramyocellular TAG content was decreased with CPT I overexpression in both the TC (-25%, P<0.05) and the EDL (-45%, P<0.05). These studies demonstrate that acute overexpression of CPT I in muscle leads to a repartitioning of FAs away from esterification and toward oxidation and highlight the importance of CPT I in regulating muscle FA metabolism.     

Hormonal and nutritional regulation of muscle carnitine palmitoyltransferase I gene expression in vivo

Transgenic mice carrying the human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene fused to a CAT reporter gene were generated to study the regulation of M-CPTI gene expression. When the mice were fasted for 48 h, CAT activity and mRNA levels increased by more than 2-fold in heart and skeletal muscle, but not liver or kidney. In the diabetic transgenic mice, there was a 2- to 3-fold increase in CAT activity and CAT mRNA levels in heart and skeletal muscle which upon insulin administration reverted to that observed with the control insulin sufficient transgenic mice. Feeding a high fat diet increased CAT activity and mRNA levels by 2- to 4-fold in heart and skeletal muscle of the transgenic mice compared to the control transgenic mice on regular diet. Overall, the M-CPTI promoter was found to be necessary for the tissue-specific hormonal and dietary regulation of the gene expression.     

Interactions of inhibitors of carnitine palmitoyltransferase I and fibrates in cultured hepatocytes.

Culture of rat hepatocytes with etomoxir, an inhibitor of carnitine palmitoyltransferase I (CPT I), for 48 h, resulted in increased carnitine acetyltransferase (CAT) activity (74%), a marked decrease in CPT activity (82%) measured in detergent extracts, and increased activities of glucose-6-phosphate dehydrogenase (227%) and fructose-1,6-bisphosphatase (65%). Changes in CAT and CPT activities were not observed after 4 h culture with etomoxir. When hepatocytes were cultured with etomoxir and benzafibrate (a hypolipidaemic analogue of clofibrate) for 48 h, etomoxir prevented the 5-fold increase in CAT activity caused by bezafibrate, whereas bezafibrate suppressed the increase in glucose-6-phosphate dehydrogenase and fructose-bisphosphatase caused by etomoxir. However, bezafibrate did not prevent the suppression of CPT activity by etomoxir. Etomoxir inhibited palmitate beta-oxidation and ketogenesis after short-term (0-4 h) and long-term (48 h) exposure, but it caused accumulation of triacylglycerol in hepatocytes only after short-term exposure (0-4 h). These effects of etomoxir on fatty acid metabolism and suppression of CPT (after 48 h) were similar in periportal and perivenous hepatocytes, but the increases in CAT and glucose-6-phosphate dehydrogenase activities were higher in periportal than in perivenous cells. The effects of CPT I inhibitors on CAT activity and long-term suppression of CPT activity are probably mediated by independent mechanisms.     

Inhibition of medium-chain fatty acid beta-oxidation in vitro by valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid

Valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid, were found to inhibit strongly the metabolism of decanoic acid in homogenates of rat liver. Reductions in decanoate consumption in response to inhibitors were paralleled by decreases in the formation of octanoic and hexanoic acids, two products of decanoate beta-oxidation. In contrast, 4-pentenoic acid, an established inhibitor of long-chain fatty acid beta-oxidation, had little effect on the metabolism of decanoate. It is concluded that the title compounds are potent, broad-spectrum inhibitors of fatty acid beta-oxidation, a property which may be of key toxicological importance in the pathology of valproate-induced liver injury.     

Evidence of a malonyl-CoA-insensitive carnitine palmitoyltransferase I activity in red skeletal muscle

Carnitine palmitoyltransferase I (CPT I), which is expressed as two distinct isoforms in liver (alpha) and muscle (beta), catalyzes the rate-limiting step in the transport of fatty acid into the mitochondria. Malonyl-CoA, a potent inhibitor of CPT I, is considered a key regulator of fatty acid oxidation in both tissues. Still unanswered is how muscle beta-oxidation proceeds despite malonyl-CoA concentrations that exceed the IC(50) for CPT Ibeta. We evaluated malonyl-CoA-suppressible [(14)C]palmitate oxidation and CPT I activity in homogenates of red (RG) and white (WG) gastrocnemius, soleus (SOL), and extensor digitorum longus (EDL) muscles. Adding 10 microM malonyl-CoA inhibited palmitate oxidation by 29, 39, 60, and 89% in RG, SOL, EDL, and WG, respectively. Thus malonyl-CoA resistance, which correlated strongly (0.678) with absolute oxidation rates (RG > SOL > EDL > WG), was greater in red than in white muscles. Similarly, malonyl-CoA-resistant palmitate oxidation and CPT I activity were greater in mitochondria from RG compared with WG. Ribonuclease protection assays were performed to evaluate whether our data might be explained by differential expression of CPT I splice variants. We detected the presence of two CPT Ibeta splice variants that were more abundant in red compared with white muscle, but the relative expression of the two mRNA species was unrelated to malonyl-CoA resistance. These results provide evidence of a malonyl-CoA-insensitive CPT I activity in red muscle, suggesting fiber type-specific expression of distinct CPT I isoforms and/or posttranslational modulations that have yet to be elucidated.     

The interaction of acyl-CoA with acyl-CoA binding protein and carnitine palmitoyltransferase I

The affinity of recombinant rat acyl-CoA binding protein (ACBP) towards acyl-CoAs was investigated using both fluorimetric analysis and isothermal titration microcalorimetry, neither of which requires the physical separation of bound and free ligand for determining the dissociation constants (K(d)). The displacement of 11-(dansylamino)undecanoyl-CoA (DAUDA-CoA) from ACBP yielded binding parameters for the competing acyl-CoAs that compared favourably with those obtained using ultra-sensitive microcalorimetric titration. The K(d) values of ACBP for oleoyl-CoA and docosahexaenoyl-CoA are 0.014 and 0.016 microM, respectively. Under identical experimental conditions, carnitine palmitoyltransferase I (CPT I) of purified rat liver mitochondria has K(d) values of 2.4 and 22.7 microM for oleoyl-CoA and docosahexaenoyl-CoA, respectively. Given that CPT I was not only present at a much lower concentration but also has an appreciably lower affinity for acyl-CoAs than ACBP, it is proposed that CPT I is capable of interacting directly with ACBP-acyl-CoA binary complexes. This is supported by the fact that the enzyme activity correlated with the concentration of ACBP-bound acyl-CoA but not the free acyl-CoA. A transfer of acyl-CoA from ACBP-acyl-CoA binary complexes to CPT I could be a result of the enzyme inducing a conformational alteration in the ACBP leading to the release of acyl-CoA.